Hydrogen peroxide is a toxin used by the human immune system to kill infectious organisms, and increasing evidence is accumulating that it is also a common second messenger in eukaryotic signaling. In humans, tumor necrosis factor, epidermal growth factor and insulin are three examples of hormones thought to signal via hydrogen peroxide. Catalase and glutathione peroxidase have long been viewed as the major nzymes degrading peroxide in cells, however, over the past few years, a distinct, highly abundant family of peroxide-reducing enzymes, peroxiredoxins (Prxs), have moved from relative obscurity to become a major Focus of redox biology research. The peroxidase activity of eukaryotic Prxs was overlooked for many years, because those Prxs that are highly expressed in eukaryotes are easily inactivated by peroxide. We have developed expertise in Prx enzymology over more than a decade of characterizing of Prxs from pathogenic bacteria (e.g. Salmonella typhimurium AhpC). These Prxs are targets for antibiotic development because of the role they play in protecting the bacteria from the human immune system. In 2003, our structural and functional studies on S. typhimurium AhpC led us to discover the structural basis for the sensitivity toward peroxides that is conserved for a subset of Prxs that are highly expressed across all eukarya. We further proposed the "floodgate hypothesis" for how this sensitivity to inactivation would actually be beneficial in organisms where hydrogen peroxide is being used as a signaling molecule, so that the antioxidant properties of the Prxs could be switched off under appropriate conditions to allow for a controlled burst in peroxide levels. Given the importance of Prxs both in pathogen defense and in human cells for combating oxidative stress and for cellular regulation, we propose here to expand our research program by both continuing our well-established work to elucidate the fundamental structural and biochemical aspects of catalysis by the variety of known Prxs (Specific Aims 1, 2, and 4) and by developing a novel systems biology approach to understand the relative contributions of Prxs and other peroxidases to peroxide homeostasis in eukaryotic cells (Specific Aim 3). Oxidative damage is thought to be important in aging, in the development of cancer and in many degenerative diseases. Moreover, impairments in cell signaling processes controlling proliferation, differentiation and apoptosis are associated with many disease states. An enhanced understanding of Prxs and the roles they play in both cell signaling and antioxidant protection will thus have important implications for the prevention of human diseases. In addition, the role of Prxs in protecting human pathogens against killing by the immune system implicates Prxs as targets for the development of new therapeutic agents to combat infectious diseases.